Qualitative and quantitative self-tests have developed gradually over the last half century. Non-instrumented tests have become commercially available using immunochemical reagents on a solid support for diagnostic tests involving HCG, LH, FSH, CKMB, Staphylococcus, and Rubella. Measurement of the hormone HCG to detect pregnancy was among the first of these tests to become commercially successful in the home market. The first home pregnancy test, the e.p.t.™, was introduced in 1977 by Warner-Lambert. The e.p.t.™ used a solution phase chemical reaction that formed a brown ring on the surface of the urine solution in the presence of HCG. The 2 hour long protocol associated with this test was sensitive to vibration and timing, causing false results.
Two additional test systems that appeared in the late 1980s were the LipoScan™ by Home Diagnostics Inc. and the Chemcard™ by Chematics Inc. Both tests measure cholesterol in whole-blood using visual color comparison. Since visual color matching is subjective, these tests do not achieve the quantitative performance necessary for cholesterol testing (Pradella et al, Clin. Chem. 36:1994-1995 (1990)).
For many analytes such as the markers for pregnancy and ovulation, qualitative or semi-quantitative tests are appropriate. There are, however, a variety of analytes that require accurate quantitation. These include glucose, cholesterol, HDL cholesterol, triglyceride, a variety of therapeutic drugs such as theophylline, vitamin levels, and other health indicators. Generally, their quantitation has been achieved through the use of an instrument. Although suitable for clinical analysis, these methods are generally undesirable for point-of-care testing in physicians offices and in the home due to the expense of the instrument.
Recently, a number of non-instrumented methods for measuring analytes use instrument-free quantitation through the use of migration distance, rather than color matching, as the visual signal. In migration distance assays, chemical/biochemical reactions occur as the analyte is wicked through a solid support. During wicking the analyte reacts with a signal-producing reagent and forms a visible signal along the support. The migration distance or the distance of signal border is related to analyte concentration. The operator reads the height of the color bar much the same way one reads a thermometer, and finds the concentration from a calibrated scale.
There are a few migration-type assays commercially available. These include Environmental Test Systems' Quantab™, which measures chloride in swimming pools and during the mixing of concrete, Syva's AccuLevel® for the measurement of therapeutic drugs, and ChemTrak's AccuMeter® for measurement of cholesterol in whole blood. Other companies such as Enzymatics and Crystal Diagnostics have more recently announced the introduction of their Q.E.D.™ and Clinimeter™ technologies to measure, respectively, alcohol in saliva and cholesterol in blood. ActiMed™ discloses a thermometer-type cholesterol assay device in Ertinghausen, U.S. Pat. No. 5,087,556 (1992).
Although these single use, thermometer-type, non-instrumented quantitative devices and non-instrumented color comparison devices for qualitative measurement have shown adequate performance, they have several problems associated with reliability and convenience. First, the colors generated on these devices are not always uniform and sharp. In the case of migration type assays the border is often light in color, unclear and difficult to read. This translates directly into user errors since the user must make a judgment related to the position of the color band border. In the case of non-instrumented pregnancy tests it is sometimes difficult to visually interpret the intensity of the colored spot (especially at HCG concentrations close to the cut-off sensitivity), and interpretation of the result is sometimes a problem. Anytime a non-technical operator is required to make a visual judgment or interpretation, an error is possible, and sometimes, is unavoidable.
Second, the assay protocol for these tests is sometimes difficult and lengthy, taking 15 minutes to 1 hour to obtain a result. Third, these tests often do not have sufficient procedural and reagent references to assure adequate test performance. Fourth and last, non-instrumented devices can only measure single endpoint type tests since enzyme rates or ratiometric analysis of two analytes cannot be measured. Therefore, the menu of potential tests is limited.
As an example of the significance of the problems, a recent article in Clinical Chemistry (Daviaud et al, Clin. Chem. 39:53-59 (1993)) evaluated all 27 home use pregnancy tests sold in France. The authors state, “among the 478 positive urine samples distributed, 230 were falsely interpreted as negative”.
In the past, immunoassays were developed for the quantitative and qualitative determination of a wide variety of compounds in a laboratory setting using detailed procedures and expensive instrumentation. Recent developments in immuno-diagnostics have resulted in a movement toward more simple approaches to the rapid analysis of clinical samples. The development of solid phase bound reagents has eliminated the need for precipitation in the separation of bound reagents from free reagents. Further advancements in solid phase immunochemistry have resulted in non-instrumented dry reagent strip immunoassays. This configuration allows for the visual qualitative or semi-quantitative determination of analytes in patient samples without the use of an instrument.
There are two basic types of non-instrumented immunoassay configurations. In the first type, or visual color zone type, a signal is generated at a specific zone on the strip where the signal indicates the presence of analyte, and the intensity of the signal indicates the concentration of the analyte in the sample. This type of assay requires visual color interpretation either for the presence of color above a threshold, as in the case of a qualitative test, or the comparison of the color intensity to a color chart, as in the case of a semi-quantitative test. In the second type, the visual signal is produced along the length of a bibulous assay strip. During wicking, the analyte reacts with a signal-producing reagent and forms a visible signal along the support. The migration distance of the signal from the proximal end of the strip is a direct measure of analyte concentration. This type of non-instrumented migration height assay can achieve quantitative results with reasonable performance as disclosed in Zuk et al, Clin. Chem. 31:1144-1150 (1985).
The color zone type of strip immunoassay is usually configured in three ways. First, a one site competitive immunoassay where labeled reagent and analyte compete for binding sites at a discrete zone along a strip where one member of the binding pair is immobilized. Second, a one site inhibition immunoassay where labeled reagent binds substantially all of the sample analyte prior to contact the strip zone where the opposite member of the binding pair is immobilized. Third, a two-site or “sandwich” immunoassay, where the sample analyte has at least two binding sites.
The prior art discloses color zone immunoassays in lateral flow and vertical flow configurations limited to the use of enzymatic signal generating systems. The use of lateral flow wicking strips has focused in the area of enzyme detection in one-site competitive or two-site sandwich configurations, and the use of particle detection has been confined largely to two-site sandwich immunoassays.
There are examples of methods developed where chemical or immunological reactions occurred along the length of a bibulous assay strip. In U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,363,537 Deutsch and Mead disclose a bibulous strip assay with discrete immunochemical reagent zones along its length for conducting specific binding assays. Grubb and Gladd, U.S. Pat. No. 4,168,146, describe an enzyme immuno-chromatography assay on a bibulous strip wherein a sample containing antigen is wicked through the assay strip, and the antigen in the sample binds to the immobilized antibody and progressively fills the binding sites as a measure of analyte concentration. The antigen containing area is visualized by wetting the strip with an enzyme labeled antibody and developing color with a chromogenic substrate. David, et al., disclose U.S. Pat. No. 4,376,110 that monoclonal antibodies with binding affinities of 108 or greater can be used in forward, reverse and simultaneous two-site sandwich immunoassays.
The lateral wicking immunoassays using colored particle detection for two-site configurations in the prior art are limited to visually interpretation and usually provide only qualitative, or at best, semi-quantitative results. The prior art fails to disclose colored particle detection in lateral wicking devices which use competitive or inhibition immunoassay configurations. Likewise, lateral wicking immunoassay reagent strips designed for use in a quantitative instrument read format are not disclosed in the prior art. Furthermore, the multiple test zone reagent strips of the prior art fail to provide quality reference.
Thus, a need exists in the field of diagnostics for a wicking assay which is sufficiently accurate and reliable to permit point-of-care use by untrained individuals in locations such as the home, sites of medical emergencies, or locations other than a clinic.